JPH0690753A - Production of hybridoma cell line - Google Patents
Production of hybridoma cell lineInfo
- Publication number
- JPH0690753A JPH0690753A JP3157731A JP15773191A JPH0690753A JP H0690753 A JPH0690753 A JP H0690753A JP 3157731 A JP3157731 A JP 3157731A JP 15773191 A JP15773191 A JP 15773191A JP H0690753 A JPH0690753 A JP H0690753A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- antibody
- cell
- hybridoma
- cell line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 75
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 139
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 30
- 239000002609 medium Substances 0.000 claims description 27
- 230000004927 fusion Effects 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
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- 229940079593 drug Drugs 0.000 claims description 9
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- 235000015097 nutrients Nutrition 0.000 claims description 2
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- 238000006386 neutralization reaction Methods 0.000 description 2
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 210000002741 palatine tonsil Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
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- 101710154606 Hemagglutinin Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 208000007514 Herpes zoster Diseases 0.000 description 1
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
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- 241001494479 Pecora Species 0.000 description 1
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- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- RIIWUGSYXOBDMC-UHFFFAOYSA-N benzene-1,2-diamine;hydron;dichloride Chemical compound Cl.Cl.NC1=CC=CC=C1N RIIWUGSYXOBDMC-UHFFFAOYSA-N 0.000 description 1
- AVVWPBAENSWJCB-DGPNFKTASA-N beta-D-galactofuranose Chemical compound OC[C@@H](O)[C@@H]1O[C@@H](O)[C@H](O)[C@H]1O AVVWPBAENSWJCB-DGPNFKTASA-N 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/166—Animal cells resulting from interspecies fusion
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明はハイブリドーマ細胞系
(セルライン)の製造法に関する。FIELD OF THE INVENTION The present invention relates to a hybridoma cell line.
(Cell line) manufacturing method.
【0002】[0002]
【従来の技術】ハイブリドーマは、雑種を形成するため
に、「不死化」細胞(特に骨髄腫細胞)を、特定物質を産生
する能力により通常選択した「正常」非形質転換細胞
(例、抗体を産生するリンパ球細胞)と融合させることに
よって形成される細胞に適用する用語である。これらの
雑種は、単一の構造および/または性質を有する物質を
産生する細胞系を得るために選択し、クローン化するこ
とができる。特に、リンパ球で形成されるかかるハイブ
リドーマは、モノクローン抗体を製造するのに使用する
ことができる。近年のハイブリドーマ技術の開発は、安
定で、取得希望の特定物質を高収率かつ特異的に産生す
る細胞系を得ることに向けられてきた。この問題につい
て各種のアプローチがなされたが、歴史的理由により、
免疫グロブリン/抗体の分野に大きく集中していた。こ
の分野での従前の活動を研究すると、直面した問題の類
別の大要と、今までに試みられた各種の解決法を概観で
きる。モノクローン性生産物を製造する雑種セルライン
の研究の最初の衝動は、生産物がモノクローン抗体であ
る免疫生物学の分野から発生した。通常の抗血清は、抗
原結合部位において構造的には異なるがそれぞれ大また
は小なる持続性および/または特異性でもって同じ抗原
に結合する非常に多数の抗体を一般に含有している。加
えてまた、通常の抗血清は、他の抗原に対するもので、
抗血清が得られる宿主個体の以前の防御反応を反映する
多数の抗体を含有している。ほとんどの目的には、かか
る広範な抗血清で充分であるが、よりすぐれた特異性や
再現性を与えることが特に診断と療法の分野において重
大な前進をもたらし、すばらしい能力をもつ科学的手段
を付与することになるであろうと考えられた。最初であ
り且つ現在では古典的な解決法は、KohlerとMilstein
がNature,256,495〜497(1975)に報告し
たが、彼らは免疫化マウス脾臓細胞を薬物感受性マウス
骨髄腫細胞に融合させることに成功した。こうして不死
化した融合細胞はインビトロで生育しそして単一細胞レ
ベルからクローン化して、均質な抗体集団(モノクロー
ン抗体)を産出する均質な細胞集団を製造することがで
きた。多くのユニークな細胞から選択し、且つ選択的な
再クローニングによって、所望の抗原特異性をもつ抗体
を産生する細胞を取得し、生育することが可能となっ
た。このような方法で製造されたマウス抗体は、研究や
診断の目的には有用であることが証明されており、また
あるものはヒトの治療にさえ用いられていた。しかし、
同様な特異性と再現性のヒト抗体がこの方法で得ること
ができるのであれば、ヒトの免疫グロブリン療法におい
て大きな前進となるであろう。またこのことは感作の危
険性を減少するであろう。BACKGROUND OF THE INVENTION Hybridomas are "normal" non-transformed cells in which "immortalized" cells (especially myeloma cells) are usually selected for their ability to produce specific substances in order to form hybrids.
The term applies to cells formed by fusing with (eg, antibody-producing lymphocyte cells). These hybrids can be selected and cloned to obtain cell lines that produce substances with a single structure and / or property. In particular, such hybridomas formed with lymphocytes can be used to produce monoclonal antibodies. Recent developments in hybridoma technology have been directed towards obtaining stable, high yielding and specific cell lines that specifically produce the particular substance of interest. Various approaches have been taken to address this issue, but for historical reasons,
It was heavily focused on the immunoglobulin / antibody field. A study of previous activities in this area gives an overview of the categorization of the problems encountered and the various solutions tried to date. The first impetus for the study of hybrid cell lines to produce monoclonal products arose from the field of immunobiology, where the product was a monoclonal antibody. Conventional antisera generally contain a large number of antibodies that structurally differ in their antigen binding sites but bind the same antigen with greater or lesser persistence and / or specificity, respectively. In addition, the usual antisera are against other antigens,
It contains a number of antibodies that reflect the previous protective response of the host individual from whom the antisera were obtained. While such a broad spectrum of antisera is sufficient for most purposes, providing greater specificity and reproducibility represents a significant advance, especially in the field of diagnostics and therapeutics, and provides a scientific tool with excellent capabilities. It was thought that it would be given. The first and now classical solutions are Kohler and Milstein
Nature, 256 , 495-497 (1975), who successfully fused immunized mouse spleen cells to drug-sensitive mouse myeloma cells. The thus immortalized fused cells could be grown in vitro and cloned from the single cell level to produce a homogeneous cell population yielding a homogeneous antibody population (monoclonal antibody). By selecting from many unique cells and selectively recloning, it has become possible to obtain and grow cells that produce antibodies with the desired antigen specificity. Mouse antibodies produced by such methods have proven useful for research and diagnostic purposes, and some have even been used in human therapy. But,
If human antibodies of similar specificity and reproducibility could be obtained in this way, it would represent a great step in human immunoglobulin therapy. This will also reduce the risk of sensitization.
【0003】インビトロでかかるヒト抗体を製造する問
題へのアプローチがいくつか試みられたが、それらは現
在のところ大きく成功していない。これらには以下のこ
とが挙げられる。 (i)Epstein−Barrウイルス(EBV)による正常なヒト
リンパ球の形質転換。このタイプの細胞は樹立するに長
くて冗長な工程を必要とし、またクローンおよび選択す
ることが非常に困難であるので、この方法はほとんど成
功しなかった。 (ii)ヒト骨髄腫細胞との正常な(ヒト)リンパ球の融合。
この方法はマウス−マウスハイブリドーマの法に明らか
に類似性を表わしているが、ヒト系ではただ一つの骨髄
腫細胞系のみしか容易に入手できないという問題に直面
し、またこの系は融合の成功を妨害するマイコプラスマ
でもって汚染されていることが判明した。 (iii)EBV−形質転換ヒトリンパ芽球様のB細胞系へ
の正常なヒトリンパ球の融合。おそらくこれはすでに報
告されたものでは最も確実で再現性ある方法であるが、
リンパ芽球様細胞が出会う基本的なインビトロの欠点を
もつ。上記細胞はクローン化することが非常に困難であ
る。またB細胞分化系統の早期段階に該当するので、そ
れらの抗体を産生し分泌する能力が真性骨髄腫のそれよ
り約10倍低い。 (iv)マウス骨髄腫へのヒトリンパ球の融合。この方法は
マウス−マウスハイブリドーマと同様に優れたインビト
ロ特性を有する細胞を製造し得るが、それは雑種が固有
の遺伝学的不安定性を有するという大きな欠点を備えて
いる。これから生ずる厄介な結果の1つは、免疫グロブ
リンの軽カッパー鎖を形成するためのゲノムが位置する
ヒト染色体を上記細胞が追放するということである。 従って、要約すれば、方法(i)は実施するにはあまりに
も冗長で非能率的である。方法(ii)は未だ実用的意義を
有していない。方法(iii)は現在利用可能なものでは最
良である。方法(iv)は実行可能であるが、細心の注意を
払うクローニング手段でもってしても生産性を維持でき
ない。以上の検討も現在までのほとんどの研究も抗体製
造に集中しているが、生産性ある細胞パートナーを、取
得希望の特定物質の分泌に関して選択し、また不死化セ
ルラインに融合できるということが明らかである。Several approaches to the problem of producing such human antibodies in vitro have been attempted, but they have so far been largely unsuccessful. These include the following: (i) Transformation of normal human lymphocytes with Epstein-Barr virus (EBV). This method has met with little success as cells of this type require long and tedious steps to establish and are very difficult to clone and select. (ii) Fusion of normal (human) lymphocytes with human myeloma cells.
Although this method represents a clear analogy to the mouse-mouse hybridoma method, it faces the problem that only one myeloma cell line is readily available in the human system, and this system does not allow successful fusion. It was found to be contaminated with disturbing mycoplasma. (iii) Fusion of normal human lymphocytes to EBV-transformed human lymphoblastoid B cell lines. Perhaps this is the most reliable and reproducible method reported previously,
It has the basic in vitro drawbacks encountered by lymphoblastoid cells. The cells are very difficult to clone. Also, since they correspond to an early stage of B cell differentiation lineage, their ability to produce and secrete antibodies is about 10 times lower than that of true myeloma. (iv) Fusion of human lymphocytes to mouse myeloma. This method can produce cells with as good in vitro properties as mouse-mouse hybridomas, but it has the major drawback that hybrids have an inherent genetic instability. One of the nasty consequences of this is that the cells expel the human chromosome in which the genome for forming the light kappa chain of the immunoglobulin is located. Therefore, in summary, method (i) is too verbose and inefficient to implement. Method (ii) has no practical significance yet. Method (iii) is the best currently available. Although method (iv) is feasible, it cannot maintain productivity even with the most careful cloning means. Although the above studies and most of the research to date have focused on antibody production, it is clear that a productive cell partner can be selected for secretion of a specific substance desired to be obtained and fused with an immortalized cell line. Is.
【0004】[0004]
【発明の記載】今回、他の細胞に更に融合させるために
親として異種間ハイブリドーマ細胞を使用することによ
り、安定性を大きく改良されたハイブリドーマを得るこ
とができるということが判明した。従って、本発明は、
不死化細胞が異種間ハイブリドーマ細胞であり、予定さ
れた物質を産生する細胞が異種間ハイブリドーマの非形
質変換パートナーと遺伝学的に適合できることを特徴と
する、上記予定物質産生細胞に融合された不死化細胞を
含むハイブリドーマセルラインの提供を可能とする。か
かる細胞の安定性は、正しく培養される場合に抗体の如
き予定された物質の産生を維持する能力をもたらす。あ
る具体例においては、不死化細胞として選択された異種
間ハイブリドーマは、免疫グロブリンを産生するそれ自
体の能力を失った骨髄腫雑種である。かかる異種間ハイ
ブリドーマの具体例は、骨髄腫細胞とリンパ球細胞の間
のものであると思われる。適切な骨髄腫細胞の具体例
は、マウスとラットから得られたものであり、免疫グロ
ブリンは産生しないものが好ましい。これらの骨髄腫は
それ自体、実際上骨髄腫の性質をもつ雑種(例、マウス
骨髄腫/マウスリンパ球)であり得る。かかる細胞は例
えばマウスSP−2骨髄種セルラインである。次いでこ
れを例えば、他の種から得られるリンパ球(例、ヒトリ
ンパ球細胞)に融合させる。好ましい実施態様では、免
疫グロブリンをもはや産生しないかかる融合で生ずる異
種間ハイブリドーマを、物質産生細胞に更に融合させる
ために選択する。これらの異種間ハイブリドーマは、染
色体喪失の点ですぐれた安定性を得るため、より一層好
適な環境を与える。不死化に使用する異種間ハイブリド
ーマは、通常の方法で更に融合する以前に薬剤耐性にす
る。方法の例は8−アザグアニン耐性に関する選択であ
る。このようにして得られるハイブリドーマは、更に融
合するための安定な親を与える。他の融合パートナー
は、予定物質を産生する能力で選択したもので(例えば
抗体の産生の場合リンパ球の選択である)、異種間ハイ
ブリドーマの非形質変換パートナーと遺伝学的に適合す
るものである。必要度の特異性を得るには、選択細胞を
あらかじめ感作して所望物質産生性にすることが望まし
い。このことは例えば抗体の場合には、特定抗原でもっ
て宿主を免疫化し次いで対応する抗体を産生する免疫細
胞(即ち、免疫適格細胞)を採集することによって達成で
きる。これらは例えば脾臓、リンパ節または血球であり
得る。DESCRIPTION OF THE INVENTION It has now been found that by using heterologous hybridoma cells as a parent for further fusion to other cells, hybridomas with greatly improved stability can be obtained. Therefore, the present invention provides
An immortalized cell fused to a predetermined substance-producing cell, characterized in that the immortalized cell is a xenogeneic hybridoma cell, and the cell producing the expected substance is genetically compatible with the non-transforming partner of the xenogeneic hybridoma. It is possible to provide a hybridoma cell line containing activated cells. The stability of such cells results in the ability to maintain the production of a predetermined substance such as an antibody when cultured correctly. In certain embodiments, the xenogeneic hybridoma selected as the immortalized cell is a myeloma hybrid that has lost its ability to produce immunoglobulins. A specific example of such a xenogeneic hybridoma appears to be between myeloma cells and lymphocyte cells. Specific examples of suitable myeloma cells are those obtained from mice and rats, preferably those which do not produce immunoglobulins. These myelomas can themselves be hybrids (eg, mouse myeloma / mouse lymphocytes) that in fact have myeloma-like properties. Such cells are, for example, the mouse SP-2 myeloma cell line. It is then fused, for example, to lymphocytes obtained from other species (eg human lymphocyte cells). In a preferred embodiment, xenogeneic hybridomas that result in such fusions that no longer produce immunoglobulin are selected for further fusion to substance producing cells. These heterologous hybridomas provide an even more favorable environment because they have excellent stability in terms of chromosome loss. The xenogeneic hybridoma used for immortalization is drug resistant prior to further fusion in the usual way. An example of a method is selection for 8-azaguanine resistance. The hybridoma thus obtained provides a stable parent for further fusion. Other fusion partners have been selected for their ability to produce the intended substance (e.g., lymphocyte selection for antibody production) and are genetically compatible with the untransformed partner of the xenogeneic hybridoma. . To obtain the required degree of specificity, it is desirable to presensitize the selected cells to produce the desired substance. For example, in the case of an antibody, this can be achieved by immunizing a host with a specific antigen and then collecting the immune cells that produce the corresponding antibody (ie, immunocompetent cells). These can be, for example, the spleen, lymph nodes or blood cells.
【0005】異種間ハイブリドーマ細胞である親の物質
産生細胞への融合は、通常の方法で行なう。これには通
常、融合を促進する物質(例、PEG1500)と共に適
当な培地で同等量の各細胞をインキュベートすることが
含まれる。所望の雑種の選択も、通常の方法でよく、選
択培地(例、不死化細胞がヒポキサンチンホスホリボシ
ル転移酵素を含有しない場合にはHAT(ヒポキサンチ
ン/アミノプテリン/チミジン))で実施することができ
る。得られるセルラインは安定で、抗体を高収率で産生
する。これらはクローン化することもでき、また要すれ
ば再選択することもできる。従って、本発明は安定なハ
イブリドーマセルラインの製造法に関し、該方法は異種
間ハイブリドーマ細胞である親を好ましくは薬剤耐性に
し、これを異種間ハイブリドーマの非形質変換パートナ
ーと遺伝学的に適合する物質産生細胞に融合させ、そし
て所望の雑種を選択することから成る。これら細胞を使
用する抗体製造は、適当な培地(例、希釈牛胎児血清を
含むもの)でインビトロでまたは宿主(例、ヌードまたは
無胸線のマウスまたはラット)への注入および腹水液の
収穫によってインビボで、通常の方法によって実施する
ことができる。方法の選択により、一つまたはそれ以上
の精製工程が必要となり得る。本発明はまた、かかる細
胞系を使用する抗体の製造を可能にする。所望の性質を
有する単純ハイブリドーマセルラインが経済的理由によ
り好ましいことは明らかであるが、所望または必要であ
れば、かかるセルラインを更に融合することも可能であ
る。本発明による典型的な抗体産生ハイブリドーマは、
親としてのヒトリンパ球と融合したマウス骨髄種を抗体
産生者としての前感作ヒトリンパ球と融合させて形成さ
れるものである。適当な場合には、骨髄種細胞自体が
(例えばマウス/マウス雑種自体であるSP−2マウス
細胞系のように)雑種であり得る。Fusion to a parent substance-producing cell, which is a cross-species hybridoma cell, is performed by a usual method. This typically involves incubating an equal volume of each cell in a suitable medium with a substance that promotes fusion (eg, PEG1500). Selection of desired hybrids may also be performed by a conventional method, and can be performed in a selective medium (eg, HAT (hypoxanthine / aminopterin / thymidine) when the immortalized cells do not contain hypoxanthine phosphoribosyltransferase). it can. The resulting cell line is stable and produces high yields of antibody. These can be cloned and, if desired, reselected. Accordingly, the present invention relates to a method for producing a stable hybridoma cell line, which method preferably renders a parent, which is a xenogeneic hybridoma cell, drug resistant, which is genetically compatible with the non-transformed partner of the xenogeneic hybridoma. Fusing to the producer cells and selecting the desired hybrid. Antibody production using these cells can be performed in vitro in a suitable medium (e.g., containing diluted fetal calf serum) or by injection into a host (e.g. nude or athymic mouse or rat) and harvest of ascites fluid. It can be carried out in vivo by conventional methods. Depending on the choice of method, one or more purification steps may be required. The invention also enables the production of antibodies using such cell lines. While it is clear that simple hybridoma cell lines with the desired properties are preferred for economic reasons, further fusion of such cell lines is possible if desired or necessary. A typical antibody-producing hybridoma according to the invention is
It is formed by fusing a mouse myeloma fused with a human lymphocyte as a parent with a presensitized human lymphocyte as an antibody producer. Where appropriate, the myeloma cells themselves
It may be a hybrid (such as the SP-2 mouse cell line which is the mouse / mouse hybrid itself).
【0006】通常の技術と従前の研究を述べている文献
の例は以下の通りである。 (1) Kohler,G.およびMilstein,C.Nature,25
6,495−497(1975) (2) Nadler,L.M.等Cancer Research,40,31
47−3154(1980) (3) Cosimi,A.B.等N,Eng1.J.Med.,305,3
05−314(1981) (4) Zurawski,V.R.等Science,199,1439−
1441(1978) (5) Koskimies,S.,Scand.J.Immunol.,11,73
−77(1980) (6) Steinitz,M.等Nature,287,443−445
(1980) (7) Olsson,L.およびKaplan,H.S.,Proc.Natl.
Acad.Sci.U,S.A.,77,5429−5431(19
80) (8) Croce,C.M.等Nature,288,488−489
(1980) (9) Nowinski,R.等Science.210,537−53
9(1980) (10) Croce,C.M.等Proc.Natl.Acad.Sci.U.S.
A.,76,3416−3419(1979) (11) Galf
re,G.等Nature266,550(1977) (12) Miller,R.A.等N.Engl.J.Med.306,51
7(1982) (13) Sikora K.,等Lancet,i 11(1982)およ
び例えば米国特許第4172124号、EP出願公開第
0043718号と0044722号。 また、Mclntyreによってモノクローン抗体に関する書
籍が最近発行されている。異種間融合の親(これは予定
物質産生能力を喪失している)を使用することによっ
て、本発明は、安定、急速な成長、クローン容易であ
り、所望物質(例、ヒト抗体)の高産生率を有するハイブ
リドーマの取得を可能にする。本発明に従って製造され
るヒト抗体は、通常の如く抗体に使用できる。かかる使
用分野の例は以下の通りである。 感染症: ウイルス(サイトロメガロウイルス、帯状痘疹
ウイルス、単純ヘルプスウイルス、肝炎AおよびBウイ
ルス、風疹ウイルス等)、バクテリア(抗毒素、抗細胞
壁)、 真菌(カンジダ)悪性疾患: 毒素の抱合ありおよびなしに
かかわらずあらゆる種類の悪性腫瘍に対する抗体 中毒: 抗毒物抗体(解毒薬、ジギタリス、オピエート
剤、三環性抗うつ剤、バルビツレート剤等) 抗イデイオタイプ: 抗・抗膵臓β細胞、抗・抗アセチル
コリンレセプター、抗リウマチ様因子 血液型抗原: 抗Rh 移植: 拒絶を抑制する抗T細胞、移植片の刺激能力を減
少する強化抗体 アレルギー: アレルゲンに対するIgG抗体、感受性減
退化の代換物 ホルモン: 避妊薬として使用する絨毛性性腺刺激ホルモ
ン抗体 ハイブリドーマ細胞自体は、免疫グロブリン遺伝子のク
ローニングを試みる場合にはmRNA源としても使用で
きる。Examples of documents describing conventional techniques and previous work are: (1) Kohler, G. and Milstein, C. Nature, 25.
6 , 495-497 (1975) (2) Nadler, LM, etc. Cancer Research, 40 , 31
47-3154 (1980) (3) Cosimi, A.B. et al N, Eng 1. J. Med., 305 , 3
05-314 (1981) (4) Zurawski, VR, et al. Science, 199 , 1439-.
1441 (1978) (5) Koskimies, S., Scand. J. Immunol., 11 , 73.
-77 (1980) (6) Steinitz, M. et al. Nature, 287 , 443-445.
(1980) (7) Olsson, L. and Kaplan, HS, Proc. Natl.
Acad. Sci. U, S.A., 77 , 5429-5431 (19)
80) (8) Croce, CM, etc. Nature, 288 , 488-489.
(1980) (9) Nowinski, R. et al. Science. 210 , 537-53.
9 (1980) (10) Croce, CM, etc. Proc. Natl. Acad. Sci. U.S.
A., 76 , 3416-3419 (1979) (11) Galf
re, G. et al. Nature 266 , 550 (1977) (12) Miller, RA. et al. N. Engl. J. Med. 306 , 51
7 (1982) (13) Sikora K., et al. Lancet, i 11 (1982) and, for example, U.S. Pat. No. 4,172,124, EP Application Publication Nos. 0043718 and 0044722. Also, a book on monoclonal antibodies has recently been published by Mclntyre. By using a heterologous fusion parent, which has lost the ability to produce the intended substance, the present invention provides stable, rapid growth, clonability, and high production of the desired substance (e.g., human antibody). Allows the acquisition of hybridomas with a certain rate. The human antibody produced according to the present invention can be used for an antibody as usual. Examples of such fields of use are as follows. Infectious diseases: Viruses (Cytolomegalovirus, Varicella zoster virus, Herpes simplex virus, Hepatitis A and B viruses, Rubella virus, etc.), Bacteria (antitoxin, anticell wall), Fungus (Candida) Malignant disease: Conjugation of toxin Antibodies against all types of malignant tumors with or without poisoning: Anti-venom antibodies (antidotes, digitalis, opiates, tricyclic antidepressants, barbiturates, etc.) Anti-idiotypes: anti / anti-pancreatic β cells, anti / anti Acetylcholine receptor, anti-rheumatoid factor Blood group antigen: Anti-Rh Transplantation: Anti-T cells that suppress rejection, enhanced antibodies that reduce the ability of the implant to stimulate the transplant Allergy: IgG antibodies to allergens, desensitizing surrogates Hormones: Contraception Chorionic gonadotropin antibody used as a drug The hybridoma cells themselves are It can also be used as a source of mRNA in the case of attempting to grayed.
【0007】本発明の特定の実施態様においては、元来
P3−X63−Ag8系と羊の赤血球細胞に対する抗体
を産生するマウス脾臓細胞のハイブリドーマ自体であっ
たSP−2細胞系であって抗体産生能力を喪失したもの
が使用される(C.F.M.Shulmann等,Nature,276,
269(1978))。これは例えばNIGMS Human
Genetic Mutant Cell Repository Ref.GM356
69Aから入手できる(US DHHS 1982 Catal
og of CellLines 参照)。このセルラインは、通常の
技術によって薬剤耐性にされ次いで正常なヒト抹消リン
パ球と融合される(C.F.G.Galfre等,Nature,26
6,550(1977)およびR.Nowinski等Science,2
10,537(1980))。多数の雑種を得、約5週間後
に急速な成長を示し且つ抗体を産生しない5クローンを
選択する。これらの細胞を8−アザグアニン耐性に関し
て選択し、かかる系の3つでもって、20μg/mlの8
−アザグアニンに耐性の変異体を得ることが可能であ
る。これらの細胞は同時にヒポキサンチン−アミノプテ
リン−チミジン(HAT)培地に感受性であり、これらが
ヒポキサンチンホスホリボシル転移酵素を産生する能力
を喪失していることを示す。これらのセルライン(SP
AZ−4)の一つを、次いで2×107SPAZ−4細胞
と108扁桃腺細胞を使用して、ヒト扁桃腺リンパ球と
インビトロで融合させる。融合は標準法に従って実施す
る。比較のために、SP/2セルラインも融合させる。
得られる細胞集団を、必要な雑種を回収するためにHA
T培地で選択する。非融合対照培養物の全細胞が死滅す
ると直ちに、HAT培地を正常な非選択性生育培地と置
きかえる。生育2週間後に抗体産生について最初の試験
を行い、各融合からの47培養体の全部がヒト抗体を産
生したことが判明する。更に4週間後に再試験すると、
SP/2ハイブリドーマの55%に対してSPAZ−4
ハイブリドーマの83%が未だに抗体を産生しているこ
とが認められる。SP/2ラインのわずか3%に対して
SPAZ−4の28%において、高い産生率が認められ
る。抗体産生喪失の主たるファクターがL鎖ゲノムの喪
失であるという事実にかんがみ、軽鎖産生の特異的試験
を行う。SPAZ−4ラインの67%がカッパー鎖、そ
の88%がラムダー鎖を生産することが認められる。S
P/2の対応する値はそれぞれ39%と61%である
(勿論、これらのパーセント値は特定の試験と使用免疫
グロブリン(1g)についてのみ比較可能である)。結果は
第1表に示す。すべての場合において、SPAZ−4が
SP/2より優れており、特に実用的見地からして最も
重要である強力な生産性に関して優れている。これらの
すべての実験を、安定なクローンに対する圧力を最大に
するために、非クローン細胞について行う(安定なクロ
ーンは多くの遺伝物質を有し、彼らに不必要な染色体の
追放に成功した細胞よりも常に成長が悪い)。しかし、
細胞をクローンするために実験を行った。現在までに得
られた結果では、安定で生産性のSP/2雑種はただ1
つも得られなかったことを示す(即ち、すべての生育細
胞が抗体を産生するセルラインはない)が得られること
を示す。他方、SPAZ−4雑種ではかかるクローンを
誘導することが可能である。本発明は、不安定性問題の
解決により、従来の公知方法よりも実質的に良好な結果
を得ることを可能にする。古典的なマウス−マウスハイ
ブリドーマが不安定であり、そしてわずらわしいサブク
ローニングが常に必要であるとしても、それが実用に供
し得ることは一言の価値がある。SPAZ−4ハイブリ
ドーマの示す不安定度はマウス−マウスハイブリドーマ
のそれよりも低いので、後者はより一層実用的でさえあ
る。In a particular embodiment of the invention, the SP-2 cell line, which was originally a hybridoma of the P3-X63-Ag8 line and a mouse spleen cell producing antibodies against sheep red blood cells, is the SP-2 cell line. Loss of capacity is used (CFM Shulmann et al., Nature, 276 ,
269 (1978)). This is for example NIGMS Human
Genetic Mutant Cell Repository Ref. GM356
69A (US DHHS 1982 Catal
og of Cell Lines). This cell line is made drug resistant by conventional techniques and then fused with normal human peripheral lymphocytes (C. F. Galfre et al., Nature, 26).
6 , 550 (1977) and R. Nowinski et al., Science, 2
10 , 537 (1980)). A large number of hybrids are obtained and 5 clones showing rapid growth and producing no antibody after about 5 weeks are selected. These cells were selected for 8-azaguanine resistance and, with three of these systems, 20 μg / ml of 8
It is possible to obtain mutants resistant to azaguanine. These cells were simultaneously sensitive to hypoxanthine-aminopterin-thymidine (HAT) medium, indicating that they have lost the ability to produce hypoxanthine phosphoribosyl transferase. These cell lines (SP
One of AZ-4) is then fused in vitro with human tonsillar lymphocytes using 2 × 10 7 SPAZ-4 cells and 10 8 tonsil cells. Fusion is performed according to standard methods. The SP / 2 cell line is also fused for comparison.
The resulting cell population was HA for recovery of the required hybrids.
Select on T medium. HAT medium is replaced with normal, non-selective growth medium as soon as all cells of the unfused control culture have died. The first test for antibody production after 2 weeks of growth reveals that all 47 cultures from each fusion produced human antibody. Retesting after another four weeks,
SPAZ-4 against 55% of SP / 2 hybridomas
It can be seen that 83% of the hybridomas are still producing antibodies. A high production rate is seen in 28% of SPAZ-4 as opposed to only 3% of the SP / 2 line. In light of the fact that the major factor in loss of antibody production is loss of the L chain genome, a specific test for light chain production is performed. It is observed that 67% of the SPAZ-4 line produces kappa chains and 88% of them produces lambda chains. S
The corresponding values of P / 2 are 39% and 61% respectively.
(Of course, these percentages are only comparable for the particular test and the immunoglobulin used (1 g)). The results are shown in Table 1. In all cases, SPAZ-4 outperforms SP / 2, especially with regard to strong productivity, which is of paramount importance from a practical standpoint. All these experiments are carried out on non-clonal cells in order to maximize the pressure on the stable clones (stable clones have more genetic material than those cells that have successfully displaced unwanted chromosomes). Always grows bad). But,
Experiments were performed to clone the cells. The results obtained to date show only one stable and productive SP / 2 hybrid
It means that none was obtained (that is, no cell line in which all the growing cells produce the antibody) was obtained. On the other hand, SPAZ-4 hybrids are capable of inducing such clones. The present invention allows the solution of the instability problem to achieve substantially better results than previously known methods. Even though the classical mouse-mouse hybridoma is unstable and always requires cumbersome subcloning, it is worthwhile to put it to practical use. The latter is even more practical because the instability of SPAZ-4 hybridomas is lower than that of mouse-mouse hybridomas.
【0008】次に実施例を挙げて本発明を具体的に説明
する。 実施例1: SPAZ−4系の製造 Ficoll−Isopaque遠心分離によって、健康な提供者の
ヘパリン化血液から末梢血液リンパ球(PBLs)を単離
する。洗浄後、pH8.0に調節したDulbeccosH21培
地50ml中の5×107SP/2細胞と108PBLsを
混合する。細胞を600×gで5分間ペレット化し、そ
の後上澄液を注意深く除去する。細胞を37℃に保ちな
がら、50%PEG4000/H21の1mlを1分で
徐々に加える。更に10mlのH21を2分で加える。
細胞を遠心分離によって集め(ペレット化)、55mlの
HAT培地に懸濁し(HAT培地: 20%胎児牛血清1
0%NCTC109、1%非必須アミノ酸、0.5%ピ
ルビン酸塩、0.2U/mlインスリン、1mMオキサル
酢酸、10-4ヒポキサンチン、4×10-7Mアミノプテ
リンおよび1.6×10-3Mチミジンを含むDulbeccos
H21)、平底組織培養マイクロプレート中の0.1ml
培養物528個に播種する。2週間毎3〜5日で新しい
HAT培地を培養物に与え、その後培地をHT培地(1
0%FCS、10-4ヒポキサンチンおよび1.6×10
-3チミジンを含むDulbeccos H21)に変える。15日
後ヒト抗体試験のために試料を取る。良好な成長を示し
抗体産生を示さない5個の培養物を選択する。次いでこ
れらを8−アザグアニン耐性サブラインの製造に選択す
る。10%FCSおよび20μg/ml8−アザグアニン
を含むDulbeccos H21に5個のセルラインを2×1
05細胞/mlで播種する。これらの培養物の3個から、
二、三週間後に生育成長する細胞を回収することができ
る。これらの1個はHATに感性のSPAZ−4であ
り、更に試験するのに使用する。Next, the present invention will be specifically described with reference to examples. Example 1: Preparation of the SPAZ-4 system Peripheral blood lymphocytes (PBLs) are isolated from heparinized blood of healthy donors by Ficoll-Isopaque centrifugation. After washing, 5 × 10 7 SP / 2 cells and 10 8 PBLs in 50 ml of Dulbeccos H21 medium adjusted to pH 8.0 are mixed. Pellet the cells at 600 xg for 5 minutes, then carefully remove the supernatant. While maintaining the cells at 37 ° C., 1 ml of 50% PEG4000 / H21 is gradually added in 1 minute. Another 10 ml of H21 are added in 2 minutes.
The cells were collected by centrifugation (pelletization) and suspended in 55 ml of HAT medium (HAT medium: 20% fetal bovine serum 1
0% NCTC109, 1% non-essential amino acids, 0.5% pyruvate, 0.2 U / ml insulin, 1 mM oxalacetic acid, 10 −4 hypoxanthine, 4 × 10 −7 M aminopterin and 1.6 × 10 −. Dulbeccos, including 3 M thymidine
H21), 0.1 ml in flat bottom tissue culture microplates
528 cultures are seeded. Fresh HAT medium is fed to the cultures every 2-5 weeks for 3-5 days, after which the medium is replaced with HT medium (1
0% FCS, 10 -4 hypoxanthine and 1.6 x 10
-Dulbeccos H21) containing -3 thymidine. Samples are taken for human antibody testing after 15 days. Five cultures are selected that show good growth and no antibody production. These are then selected for the production of 8-azaguanine resistant sublines. Dulbeccos H21 containing 10% FCS and 20 μg / ml 8-azaguanine, 5 cell lines 2 × 1
Seed at 0 5 cells / ml. From three of these cultures,
Cells that grow and grow after a few weeks can be collected. One of these is HAT-sensitive SPAZ-4 and is used for further testing.
【0009】実施例2: 扁桃腺リンパ球との融合 小児から扁桃腺を結合組織部で切り取り、微細金属網を
通過させて、単一細胞プレパラートを得る。赤血球の数
を減ずるために、全細胞をFicoll−Isopaqueで分画す
る。得られる細胞集団の108の細胞を、実施例1で述
べたのと同じ方法でもって2×107のSPAZ−4ま
たはSP/2細胞に融合させる。その後細胞をHAT培
地の培養物0.5mlの47個に播種し、この時にシクロ
スポリンA0.5μg/mlを補給する。1日後にシクロ
スポリンAを含まないHAT培地0.5mlを加える。3
日目にすでに培地の50%がHT培地に変化する。この
後毎3〜5日の経過でもって細胞をHT培地に維持す
る。 免疫グロブリン製造試験 従来のELISA(固相酵素免疫検定)システムを使用す
る。pH9.6の重炭酸塩緩衝剤中1:400の希釈でウ
サギ抗ヒト免疫グロブリンをNunc EIAプレートに捕
捉させる。該プレートを洗浄後、培養上清を37℃で3
0分インキュベートする。再度洗浄後、1:400の希
釈でペルオキシダーゼ抱合ウサギ抗ヒトIgG、IgM、
IgA試剤(Miles−Yeda)でインキュベーション物を処
理する。洗浄後酵素反応物を1,2−フェニレンジアミ
ン二塩酸塩とH2O2で発色させる。ヤギ抗ヒトラムダー
またはヤギ抗ヒトカッパー試剤(Tago)を1:3000の
希釈でウサギ抗ヒトIgG、IgM、IgAの代わりに使
用する以外は同じ方法で、軽鎖産生試験を行う。結果を
Titertek Multiscan Elisa 光度計で評価し、第1表
の「4」と「7」の値をこの光度計からの低い読みと高い読
みとしてそれぞれ表示する。Example 2: Fusion with tonsillar lymphocytes Tonsils are cut from a child at the connective tissue part and passed through a fine metal mesh to obtain a single cell preparation. Whole cells are fractionated with Ficoll-Isopaque to reduce the number of red blood cells. 10 8 cells of the resulting cell population are fused to 2 × 10 7 SPAZ-4 or SP / 2 cells in the same manner as described in Example 1. The cells are then seeded in 47 of a 0.5 ml culture of HAT medium, at which time cyclosporin A 0.5 μg / ml is supplemented. One day later, 0.5 ml of HAT medium without cyclosporin A is added. Three
Already on day 50% of the medium is changed to HT medium. After this, the cells are maintained in HT medium every 3-5 days. Immunoglobulin production test A conventional ELISA (solid phase enzyme immunoassay) system is used. Rabbit anti-human immunoglobulin is captured on Nunc EIA plates at a dilution of 1: 400 in bicarbonate buffer at pH 9.6. After washing the plate, the culture supernatant was incubated at 37 ° C for 3
Incubate for 0 minutes. After washing again, a 1: 400 dilution of peroxidase-conjugated rabbit anti-human IgG, IgM,
Incubate with IgA reagent (Miles-Yeda). After washing, the enzyme reaction is developed with 1,2-phenylenediamine dihydrochloride and H 2 O 2 . Light chain production studies are performed in the same manner except that goat anti-human lambda or goat anti-human kappa reagent (Tago) is used in place of rabbit anti-human IgG, IgM, IgA at a 1: 3000 dilution. The results are evaluated with a Titertek Multiscan Elisa photometer and the "4" and "7" values in Table 1 are displayed as the low and high readings from this photometer, respectively.
【0010】実施例3: インフルエンザAおよびB型に
対するモノクローン抗体の製造(インビボ免疫後) 使用したインフルエンザワクチンは、A/バンコック、
A/ブラジルおよびB/シンガポールからの赤血球凝集
素とノイラミニダーゼを含む商品名SANDOVACで
ある。3人の健康な志願者を免疫化し、3、7、10、
14および17日に採血する。各回50〜60mlの血
液をひじ静脈からヘパリン含有注射器へ抜く。リンパ球
をFicoll−Paque(Pharmacia)上の遠心分離によって
単離し、使用前に塩水で2回洗う。融合の親は a) SPAZ−4(実施例1参照) b) SP/2(上記参照) c) WISTAR INSTITUTEで8−アザグア
ニン耐性にしたGM1500ヒトリンパ芽球様細胞(ヒ
トIgG2カッパーを産生する) である。SPAZ−4とSP/2は全く同じに融合す
る。骨髄腫(107細胞)とヒトリンパ球(約3×107細
胞)を、pH8.0でDMEM無血清培地の存在下試験管
で混合する。細胞を600×gで5分遠心分離にかけ
る。得られるペレットを50%PEG4000で1分処
理し、その後PEGを培地で徐々に希釈する。1回洗浄
後、20%胎児牛血清を含むDMEM−HAT培地の1
00μl培養物176個に細胞を播種する。細胞を湿気
のある10%CO2−大気で37℃で培養する。GM
1500細胞(107細胞)をpH8.0の無血清DMEM
中でヒトリンパ球(約3×107細胞)と混合し、600
×gで5分遠心分離する。ペレットを50%PEG60
00で5分処理し、その間に細胞を600×gで遠心分
離する。この後PEGを培地で徐々に希釈する。1回洗
浄後、20%胎児牛血清を含むRPMI 1640−H
AT培地の100μl培養物176個に細胞を播種す
る。細胞を湿気のある5%CO2−大気で37℃で培養
する。6160個の培養物(GM1500により264
0体、SPAZ−4により2464体、SP/2により
1056体)を行ない全体で35個の融合(GM1500
に15体、SPAZ−4に14体、SP/2に6体)を
得る。一般に毎3〜5日間で、細胞増殖が必要とすると
きはいつでも、培地を培養物に変える。上澄液に抗イン
フルエンザ抗体が存在することをELISA法で検定す
る。関連インフルエンザ抗原をマイクロプレートウエル
(Nunc)に捕捉し、上澄液を加え、抗原と相互作用させ
る。洗浄後ぺルオキシダーゼ抱合ウサギ抗ヒトIgG、
IgA、IgM(Miles)をウエルに加える。インキュベー
ション後非結合ペルオキシダーゼ抱合体を洗浄除去し、
酵素発色物質をウエルに加える。反応を視覚で評価し、
Titertekマイクロプレート光度計で証明する。陽性結
果を示す培養物を、マウス胸線細胞供給細胞と共に10
0μlのDMEM+20%胎児牛血清中に(SPAZ−
4およびSP/2由来細胞)、またはMRC−5供給細
胞のRPMI 1640+20%胎児牛血清中にGM
1500由来細胞)、1細胞/培養物で細胞を播種する
88個の新しいウエルに限界希釈条件でクローン化す
る。生産性細胞は大スケールで生育させ、液体N2中に
凍結する。合計86個の培養物をクローン化した(GM
1500から58体、SPAZ−4から26体、SP
/2から2体)。細胞をクローン化すべきであるか否か
については、その決定に全く自由な基準を適用するがこ
れはクローン化後に真に陽性の細胞の収率が低い結果を
招き得る。。例えばGM 1500は高産生細胞を製造
しないのでこの決定が必要となる。SPAZ−4細胞か
らは4個の陽性クローンが確認され、SP/2からは1
個が確認されるが、しかし後者は雑種ではなく、自然に
生起したEBウイルス形質変換細胞であった。産生細胞
はGM 1500からは誘導されない。融合の結果を下
記表に要約して示す。Example 3: Production of monoclonal antibodies against influenza A and B (after in vivo immunization) The influenza vaccine used was A / Bangkok,
Trade name SANDOVAC containing hemagglutinin and neuraminidase from A / Brazil and B / Singapore. Immunize 3 healthy volunteers with 3, 7, 10,
Blood is drawn on days 14 and 17. 50-60 ml of blood is drawn from the elbow vein each time into a heparin-containing syringe. Lymphocytes are isolated by centrifugation on Ficoll-Paque (Pharmacia) and washed twice with saline before use. The parent of the fusion is a) SPAZ-4 (see Example 1) b) SP / 2 (see above) c) 8-azaguanine resistant GM1500 human lymphoblastoid cells (producing human IgG2 kappa) with WISTAR INSTITUTE. is there. SPAZ-4 and SP / 2 fuse exactly the same. Myeloma (10 7 cells) and human lymphocytes (about 3 × 10 7 cells) are mixed in vitro in the presence of DMEM serum-free medium at pH 8.0. The cells are centrifuged at 600 xg for 5 minutes. The resulting pellet is treated with 50% PEG4000 for 1 minute, after which the PEG is gradually diluted with medium. After washing once, 1 of DMEM-HAT medium containing 20% fetal bovine serum
Cells are seeded in 176 00 μl cultures. The cells are incubated at 37 ° C. in a humidified 10% CO 2 atmosphere. GM
1500 cells (10 7 cells) were used in serum-free DMEM having a pH of 8.0.
Mixed with human lymphocytes (about 3 × 10 7 cells) in
Centrifuge at xg for 5 minutes. 50% PEG60 pellet
Treatment with 00 for 5 minutes, during which the cells are centrifuged at 600 xg. After this, PEG is gradually diluted with medium. After washing once, RPMI 1640-H containing 20% fetal bovine serum
Cells are seeded in 176 100 μl cultures of AT medium. The cells are incubated at 37 ° C. in a humidified 5% CO 2 atmosphere. 6160 cultures (264 with GM 1500
0, 2464 by SPAZ-4, 1056 by SP / 2) and 35 fusions (GM1500)
In SPAZ-4, 14 in SPAZ-4, and 6 in SP / 2). Generally every 3-5 days, the medium is changed to a culture whenever cell growth requires. The presence of anti-influenza antibody in the supernatant is assayed by the ELISA method. Related influenza antigens in microplate wells
Capture in (Nunc) and add supernatant to interact with antigen. After washing, peroxidase-conjugated rabbit anti-human IgG,
Add IgA, IgM (Miles) to wells. After incubation, wash away unbound peroxidase conjugate,
Enzyme chromogen is added to the wells. Evaluate the reaction visually,
Prove with Titertek microplate photometer. Cultures showing positive results were used in combination with mouse pleural cell feeder cells for 10
In 0 μl DMEM + 20% fetal bovine serum (SPAZ-
4 and SP / 2-derived cells), or MRC-5-supplied cells in RPMI 1640 + 20% fetal bovine serum.
1500 derived cells), cloned in limiting dilution conditions into 88 new wells seeding cells at 1 cell / culture. Productive cells are grown on a large scale and frozen in liquid N 2 . A total of 86 cultures were cloned (GM
1,500 to 58 bodies, SPAZ-4 to 26 bodies, SP
/ 2 to 2). Whether or not cells should be cloned applies completely free criteria in its determination, which can result in low yields of truly positive cells after cloning. . This determination is necessary because, for example, GM 1500 does not produce high producing cells. Four positive clones were confirmed from SPAZ-4 cells, and 1 from SP / 2.
However, the latter was not a hybrid but a naturally occurring EB virus-transformed cell. Producer cells are not derived from GM 1500. The results of the fusion are summarized in the table below.
【0011】[0011]
【表1】 [Table 1]
【0012】5個の陽性セルラインを数回再クローン
し、大スケールで成長させる。これはEBV系の消滅を
もたらす(かかる細胞コロニーが低密度クローニングを
生存させないことはよく知られている)。4個の陽性S
PAZ−4ハイブリドーマセルラインをC15、C2
8、C29およびC75として確認した。Five positive cell lines are recloned several times and grown on a large scale. This results in the disappearance of the EBV system (it is well known that such cell colonies do not survive low density cloning). 4 positive S
PAZ-4 hybridoma cell line is C15, C2
8, C29 and C75.
【0013】実施例4: 抗インフルエンザハイブリドー
マの製造(インビトロ免疫後) ヒト脾臓細胞をFalcon2051チューブ中の2mlに1
06/mlで播種し、全体で34℃106の細胞を使用す
る。最適量の庶糖濃度勾配精製A/バンコックウイルス
を加え、培養物を5%ヒト熱不活化血漿を含むRPMI
1640培地で空気中5%CO2の雰囲気下37℃で1
01時間維持する。回収した9×106細胞を実施例3
に述べたのと同様にして同数のSPAZ−4細胞に融合
する。細胞を176個の培養物に播種し、20%胎児牛
血清、HATおよび1μg/mlシクロスポリンAを含む
DulbeccosのMEMで成長させる。2日後HAT培地を
HT培地で次第に置換し、その後4〜5日の間隔で変え
る。融合後12日と19日目に培養物を抗インフルエン
ザ抗体についてスクリーニングし、陽性培養物をクロー
ン化する。陽性クローン(B2として確認)を得、これは
大スケールで成長させることができ、また薬剤用の純粋
抗体をインビトロで製造するのに使用することができ
る。Example 4: Preparation of anti-influenza hybridoma (after in vitro immunization) Human spleen cells were added to every 2 ml in Falcon 2051 tube.
Seed at 0 6 / ml and use cells at 34 ° C 10 6 in total. RPMI containing 5% human heat-inactivated plasma with the addition of an optimal amount of sucrose gradient A / Bangkok virus.
1640 medium at 37 ° C. in an atmosphere of 5% CO 2 in air 1
Hold for 01 hour. The collected 9 × 10 6 cells were used in Example 3
Are fused to an equal number of SPAZ-4 cells as described above. Cells are seeded in 176 cultures and grown in Dulbeccos MEM containing 20% fetal bovine serum, HAT and 1 μg / ml cyclosporin A. After 2 days the HAT medium is gradually replaced with HT medium and then changed at intervals of 4-5 days. Cultures are screened 12 days and 19 days post fusion for anti-influenza antibodies and positive cultures are cloned. A positive clone (identified as B 2 ) was obtained, which can be grown on a large scale and can be used to make pure antibodies for drugs in vitro.
【0014】実施例5: ヒト抗インフルエンザモノクロ
ーン抗体の特性確認 a) 抗体/C15 この抗体はIgG1クラスのものであり、カッパー軽鎖
を有する。抗体は試験したすべてのインフルエンザA型
ウイルス(H1N1、H2N2、H3N2)に反応し、そ
れは恐らくウイルスの核タンパクに向けられている。抗
体はインビトロで中和活性を有せず、またインビボで保
護効果を有していない。 b) 抗体/C28 この抗体はIgG1クラスのものであり、ラムダー軽鎖
を有している。抗体はH3N2型のインフルエンザウイ
ルスに反応するのみであり、それは恐らくウイルスの赤
血球凝集素に向けられている。抗体はインビトロで強い
中和効果を有し、またインビボで劇的な保護活性を有し
ている。それは試験したすべてのH3N2ウイスル(即
ち、1968、1969、1973、1974、197
5、1977、1979および1980からのウイル
ス)を中和することができる。MDCK細胞上の197
3A/ポートチャーマーズで試験すると、純粋抗体1.
5mg/mlを含む抗体製品は、中和価12800を有
することが認められる。同一実験において、標準ヒト免
疫グロブリン製剤(Sandoglobulin)は濃度60mg/m
lで中和価50を有することが認められる。このこと
は、モノクローン抗体C28がタンパク質当量レベルで
正常な免疫グロブリンよりも10240倍高い能力を有
していることを意味している。これに関連して、抗イン
フルエンザ抗体が正常な免疫グロブリン製剤の越的成分
一つであるということを指摘したい。このことは、C2
8と同じ中和能力を有し、例えばサイトメガロウウイル
スまたは帯状痘疹ウイルス(この場合正常な免疫グロブ
リン製剤の抗体レベルはかなり低い)に対する抗体は、
相対的能力がより一層高い値に達することを意味する。 c) 抗体/C29 この抗体はIgG1クラスのものであり、ラムダー軽鎖
を有している。抗体はB/シンガポール型インフルエン
ザに反応する。他のB型ウイルスに対する試験は行わな
かった。A型ウイルスに対しては活性を有していない。 d) 抗体/C75 この抗体はIgG3クラスのものであり、カッパー軽鎖
を有している。これはH3N2型ウイルスに反応するの
みであり、恐らくはウイルスの赤血球凝集素に向けられ
ている。これはインビトロで中和活性を有せず、インビ
ボで保護効果を有しない。 e) 抗体/B2 この抗体はIgG1クラスのものであり、カッパー軽鎖
を有している。これはH3N2型ウイルスに反応するの
みであり、恐らくはウイルスの赤血球凝集素に向けられ
ている。抗体はインビトロで中和効果を有せず、インビ
ボ保護試験は行わなかった。Example 5: Characterization of human anti-influenza monoclonal antibody a) Antibody / C15 This antibody is of the IgG1 class and has a kappa light chain. The antibody reacts with all influenza A viruses tested (H1N1, H2N2, H3N2), which are probably directed against the viral nucleoprotein. The antibody has no neutralizing activity in vitro and has no protective effect in vivo. b) Antibody / C28 This antibody is of the IgG1 class and has a lambda light chain. The antibody only reacts to the H3N2 influenza virus, which is probably directed against the viral hemagglutinin. Antibodies have a strong neutralizing effect in vitro and have a dramatic protective activity in vivo. It tested all H3N2 viruses (ie 1968, 1969, 1973, 1974, 197).
5, 1977, 1979 and 1980). 197 on MDCK cells
Pure antibody when tested at 3A / Port Charmers 1.
The antibody product containing 5 mg / ml is found to have a neutralization number of 12800. In the same experiment, the standard human immunoglobulin preparation (Sandoglobulin) had a concentration of 60 mg / m 2.
It is found to have a neutralization number of 50 at 1 This means that the monoclonal antibody C28 is 10240 times more potent than normal immunoglobulin at the protein equivalent level. In this connection, I would like to point out that anti-influenza antibodies are one of the transcendental components of normal immunoglobulin preparations. This is C2
An antibody with the same neutralizing capacity as 8, eg against cytomegalovirus or herpes zoster virus (wherein normal immunoglobulin preparations have significantly lower antibody levels),
It means that the relative capacity reaches an even higher value. c) Antibody / C29 This antibody is of the IgG1 class and has a lambda light chain. The antibody reacts with B / Singapore influenza. No tests were performed against other type B viruses. It has no activity against type A virus. d) Antibody / C75 This antibody is of the IgG3 class and has a kappa light chain. It only responds to the H3N2 virus and is probably directed to the viral hemagglutinin. It has no neutralizing activity in vitro and no protective effect in vivo. e) Antibody / B2 This antibody is of the IgG1 class and has a kappa light chain. It only responds to the H3N2 virus and is probably directed to the viral hemagglutinin. The antibody had no neutralizing effect in vitro and was not tested in vivo.
【0015】[0015]
【表2】 [Table 2]
【0016】本発明により実施可能となるものを列挙す
ると次の通りである。 1.不死化細胞が異種間ハイブリドーマ細胞であり、予
定物質産生細胞が異種間ハイブリドーマの非形質転換パ
ートナーと遺伝学的に適合することを特徴とする、上記
予定物質産生細胞に融合した不死化細胞を含むハイブリ
ドーマセルライン。 2.物質産生細胞が異種間ハイブリドーマ細胞母体の非
形質転換パートナーと同一種のものである上記第1項記
載のハイブリドーマセルライン。 3.物質産生細胞パートナーと、異種間ハイブリドーマ
細胞母体の非形質転換パートナーがヒト起源のである上
記第2項記載のハイブリドーマセルライン。 4.産生物質が抗体である上記第1〜3項のいずれか1
項記載のハイブリドーマセルライン。 5.物質産生細胞が予定物質を産生するようにインビト
ロまたはインビボで前感作されている上記第4項記載の
ハイブリドーマセルライン。 6.物質産生細胞がリンパ球である上記第1〜5項のい
ずれか1項記載のハイブリドーマセルライン。 7.リンパ球がヒト起源のものである上記第6項記載の
ハイブリドーマセルライン。 8.リンパ球が抗体を産生するようにインビボまたはイ
ンビトロであらかじめ感作されている上記第6または第
7項記載のハイブリドーマセルライン。 9.異種間ハイブリドーマ細胞の骨髄腫パートナーが免
疫グロブリンを産生しないネズミ骨髄腫である上記第1
〜8項のいずれか1項記載のハイブリドーマセルライ
ン。 10.ネズミ骨髄腫がマウス骨髄腫である上記第9項記載
のハイブリドーマセルライン。 11.マウス骨髄腫がSP−2細胞系によってもたらされ
る上記第10項記載のハイブリドーマセルライン。 12.異種間ハイブリドーマ細胞母体が予定物質を産生す
る能力を失っているかまたは他の理由で産生することが
できない上記第1〜11項のいずれか1項記載のハイブ
リドーマセルライン。 13.マウス骨髄腫細胞と親細胞としてのヒトリンパ球の
融合による異種間ハイブリドーマ細胞および物質産生細
胞としてのあらかじめ感作された抗体産生ヒトリンパ球
を含む上記第1〜12項のいずれかのハイブリドーマセ
ルライン。 14.異種間ハイブリドーマ細胞を薬剤耐性にし、これを
異種間ハイブリドーマの非形質転換パートナーと遺伝学
的に適合する物質産生細胞に融合させ、そして所望の雑
種を選択することを特徴とする安定なハイブリドーマセ
ルラインの製造法。 15.使用する融合パートナーを上記第1〜13項のいず
れかに述べたものから選択する上記第14項記載の方
法。 16.薬剤耐性を8−アザグアニンに耐性の細胞から選択
することによって達成する上記第14または15項記載
の方法。 17.融合をこれを促進する物質の存在下に行う上記第1
4〜16項のいずれか1項記載の方法。 18.促進物質がポリエチレングリコールである上記第1
7項記載の方法。 19.所望雑種の選択を、HAT感受性の欠如と予定物質
産生能力の検定を基礎として行う上記第14〜18項の
いずれか1項記載の方法。 20.異種間ハイブリドーマ細胞である親と物質産生細胞
を、これら細胞の融合促進剤と共に栄養培養培地に含む
ことから成る細胞融合システム。 21.細胞パートナーが上記第1〜13項のいずれかに述
べられているものである上記第20項記載の細胞融合シ
ステム。 22.融合促進剤がポリエチレングリコールである上記第
20または21項記載の細胞融合システム。 23.上記第1〜13項のいずれかのまたは上記第14〜
19項のいずれかに従って製造されたハイブリドーマセ
ルラインをそのためのインビトロまたはインビボ培養培
地で培養し、その後該培地から予定物質を単離すること
を特徴とする該物質の製造法。 24.得られる物質が抗体である上記第23項記載の方
法。 25.インビボ培養を無胸線(ヌード)マウスまたはラット
中で行う上記第23または24項記載の方法。 26.ハイブリドーマセルラインを製造するために細胞融
合においてパートナーとして使用するものである上記第
9〜13項のいずれか1項記載の異種発生性ハイブリド
ーマセルライン。 27.マウス/ヒト/ヒトハイブリドーマセルライン。 28.予定の特異性を有するヒト抗体を産生可能な安定な
マウス/ヒト/ヒトハイブリドーマセルライン。 29.マウス細胞がマウス/マウスハイブリドーマ自体で
ある上記第27または28項のハイブリドーマセルライ
ン。 30.抗体産生能力を喪失している薬剤耐性のマウス/ヒ
トハイブリドーマを製造し、別のヒト染色体に好都合な
環境を与え且つ抗体産生維持に高安定性を示すヒト化マ
ウス骨髄腫細胞を構築することを特徴とする、長期間に
わたってヒト抗体を産生するセルラインの製造法。 31.通常の方法でSP−2セルラインを正常なヒトリン
パ球と融合させ、抗体産生なしに急速成長を示す取得雑
種からクローンを選択し、これを更に8−アザグアニン
耐性に関して選択し、これを再びヒトリンパ球と融合さ
せ、そしてHAT培地中で得られた細胞集団を選択する
ことを特徴とする上記第30項記載の方法。The items that can be implemented by the present invention are listed below. 1. An immortalized cell fused to a predetermined substance-producing cell, characterized in that the immortalized cell is a xenogeneic hybridoma cell and the predetermined substance-producing cell is genetically compatible with a non-transforming partner of the xenogeneic hybridoma Hybridoma cell line. 2. The hybridoma cell line of claim 1, wherein the substance-producing cells are of the same species as the non-transforming partner of the heterologous hybridoma cell matrix. 3. The hybridoma cell line according to the above item 2, wherein the substance producing cell partner and the non-transforming partner of the heterologous hybridoma cell matrix are of human origin. 4. Any one of the above items 1 to 3, wherein the produced substance is an antibody
The hybridoma cell line described in the above item. 5. The hybridoma cell line of claim 4, wherein the substance-producing cells have been presensitized in vitro or in vivo to produce the intended substance. 6. The hybridoma cell line according to any one of the above items 1 to 5, wherein the substance-producing cells are lymphocytes. 7. The hybridoma cell line of claim 6, wherein the lymphocytes are of human origin. 8. The hybridoma cell line of claim 6 or 7, wherein the lymphocytes have been presensitized in vivo or in vitro to produce antibodies. 9. First, the myeloma partner of the xenogeneic hybridoma cells is a murine myeloma that does not produce immunoglobulin
9. The hybridoma cell line according to any one of items 8 to 8. Ten. The hybridoma cell line of claim 9, wherein the murine myeloma is a mouse myeloma. 11. The hybridoma cell line of claim 10, wherein the mouse myeloma is produced by the SP-2 cell line. 12. The hybridoma cell line according to any one of the above items 1 to 11, wherein the heterologous hybridoma cell matrix has lost the ability to produce a predetermined substance or cannot be produced for other reasons. 13. 13. The hybridoma cell line according to any one of the above items 1 to 12, which comprises cross-species hybridoma cells obtained by fusing mouse myeloma cells and human lymphocytes as parent cells and presensitized antibody-producing human lymphocytes as substance-producing cells. 14. A stable hybridoma cell line characterized by rendering a xenogeneic hybridoma cell drug resistant, fusing it to a substance producing cell that is genetically compatible with the non-transforming partner of the xenogeneic hybridoma, and selecting the desired hybrid. Manufacturing method. 15. The method according to claim 14, wherein the fusion partner to be used is selected from those described in any of the above items 1 to 13. 16. 16. The method according to the above 14 or 15, wherein the drug resistance is achieved by selecting from cells resistant to 8-azaguanine. 17. The first, wherein the fusion is carried out in the presence of a substance promoting this.
The method according to any one of 4 to 16. 18. The above first in which the accelerating substance is polyethylene glycol.
The method according to item 7. 19. The method according to any one of the above items 14 to 18, wherein the desired hybrid is selected on the basis of the lack of HAT sensitivity and the assay of the ability to produce a predetermined substance. 20. A cell fusion system comprising a parental culture medium which is a cross-species hybridoma cell and a substance producing cell together with a fusion promoter for these cells in a nutrient culture medium. twenty one. Item 21. The cell fusion system according to Item 20, wherein the cell partner is one described in any one of Items 1 to 13. twenty two. 22. The cell fusion system according to the above 20 or 21, wherein the fusion promoter is polyethylene glycol. twenty three. Any of the above 1 to 13 or the above 14 to
20. A method for producing a hybridoma cell line produced according to any of paragraphs 19 to 19 by culturing it in an in vitro or in vivo culture medium therefor and then isolating the desired substance from the medium. twenty four. 24. The method according to the above item 23, wherein the obtained substance is an antibody. twenty five. 25. The method according to the above 23 or 24, wherein the in vivo culture is performed in athymic (nude) mice or rats. 26. A heterogeneous hybridoma cell line according to any one of paragraphs 9 to 13 above, which is used as a partner in cell fusion to produce a hybridoma cell line. 27. Mouse / human / human hybridoma cell line. 28. A stable mouse / human / human hybridoma cell line capable of producing a human antibody having a predetermined specificity. 29. The hybridoma cell line of paragraph 27 or 28, wherein the mouse cell is the mouse / mouse hybridoma itself. 30. To produce drug-resistant mouse / human hybridomas that have lost antibody-producing ability, and to construct humanized mouse myeloma cells that provide a favorable environment for another human chromosome and exhibit high stability in maintaining antibody production. A method for producing a cell line that produces a human antibody for a long period of time. 31. The SP-2 cell line was fused with normal human lymphocytes in the usual way, and a clone was selected from the obtained hybrids that showed rapid growth without antibody production, which was further selected for 8-azaguanine resistance, which was again human lymphocyte. 31. A method according to claim 30, characterized in that the cell populations which are fused with and obtained in HAT medium are selected.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 (C12N 5/28 C12R 1:91) (C12P 21/08 C12R 1:91) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI technical display location (C12N 5/28 C12R 1:91) (C12P 21/08 C12R 1:91)
Claims (3)
ブリドーマの元になった形質転換されていないパートナ
ー細胞と遺伝学的に適合する目的抗体産生リンパ球に融
合させ、そして目的とする雑種を選択することを特徴と
する、安定なハイブリドーマセルラインの製造法。1. Fusing a xenogeneic hybridoma cell with an antibody-producing lymphocyte of interest that is genetically compatible with the untransformed partner cell from which the xenogeneic hybridoma was derived, and selecting the hybrid of interest. A method for producing a stable hybridoma cell line, which comprises:
融合させる前に薬剤耐性にする、特許請求の範囲第1項
記載の方法。2. The method of claim 1 wherein the xenogeneic hybridoma cells are drug resistant prior to fusing with lymphocytes.
ンパ球を、これら細胞の融合促進剤と共に栄養培養培地
中に含む細胞融合培地。3. A cell fusion medium comprising a heterologous hybridoma cell, parent and lymphocyte, in a nutrient culture medium together with a fusion promoter for these cells.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CH409/82 | 1982-01-22 | ||
CH409/82A CH652145A5 (en) | 1982-01-22 | 1982-01-22 | METHOD FOR IN VITRO PRODUCTION OF HYBRID OMEN WHAT human monoclonal antibodies GENERATE. |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP58009241A Division JPS58128323A (en) | 1982-01-22 | 1983-01-21 | Manufacture of human monochrone antibody |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0690753A true JPH0690753A (en) | 1994-04-05 |
JPH0789909B2 JPH0789909B2 (en) | 1995-10-04 |
Family
ID=4186382
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP58009241A Granted JPS58128323A (en) | 1982-01-22 | 1983-01-21 | Manufacture of human monochrone antibody |
JP2267500A Granted JPH03236794A (en) | 1982-01-22 | 1990-10-03 | Preparation of human monoclonal antibody |
JP3157731A Expired - Lifetime JPH0789909B2 (en) | 1982-01-22 | 1991-05-30 | Manufacturing method of hybridoma cell line |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP58009241A Granted JPS58128323A (en) | 1982-01-22 | 1983-01-21 | Manufacture of human monochrone antibody |
JP2267500A Granted JPH03236794A (en) | 1982-01-22 | 1990-10-03 | Preparation of human monoclonal antibody |
Country Status (17)
Country | Link |
---|---|
US (1) | US4634664A (en) |
JP (3) | JPS58128323A (en) |
AT (1) | AT388932B (en) |
CH (1) | CH652145A5 (en) |
CY (1) | CY1488A (en) |
DE (1) | DE3301249A1 (en) |
DK (1) | DK23183A (en) |
FI (1) | FI83538C (en) |
FR (1) | FR2522679B1 (en) |
GB (1) | GB2113715B (en) |
HK (1) | HK52589A (en) |
IL (1) | IL67721A (en) |
IT (1) | IT1160468B (en) |
KE (1) | KE3884A (en) |
MY (1) | MY8700169A (en) |
SE (1) | SE502344C2 (en) |
SG (1) | SG28189G (en) |
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JPH03236794A (en) | 1991-10-22 |
HK52589A (en) | 1989-07-07 |
ATA19583A (en) | 1989-02-15 |
FI830190A0 (en) | 1983-01-20 |
FI830190L (en) | 1983-07-23 |
MY8700169A (en) | 1987-12-31 |
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DE3301249C2 (en) | 1990-04-05 |
IL67721A0 (en) | 1983-05-15 |
CH652145A5 (en) | 1985-10-31 |
DK23183D0 (en) | 1983-01-20 |
FR2522679B1 (en) | 1986-05-09 |
SE502344C2 (en) | 1995-10-09 |
FI83538C (en) | 1991-07-25 |
JPS58128323A (en) | 1983-07-30 |
SG28189G (en) | 1989-08-11 |
IT8319234A0 (en) | 1983-01-21 |
JPH0789909B2 (en) | 1995-10-04 |
FR2522679A1 (en) | 1983-09-09 |
JPH0471520B2 (en) | 1992-11-13 |
DK23183A (en) | 1983-07-23 |
CY1488A (en) | 1989-12-08 |
IL67721A (en) | 1986-04-29 |
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